The following is a description of relevant art, none of which is admitted to be prior art to the claims.
Glutamate is the major excitatory neurotransmitter in the mammalian brain. Glutamate binds or interacts with one or more glutamate receptors which can be differentiated pharmacologically into several subtypes. In the mammalian central nervous system (CNS) there are three main subtypes of ionotropic glutamate receptors, defined pharmacologically by the selective agonists N-methyl-D-aspartate (NMDA), kainate (KA), and .alpha.-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). The NMDA receptor has been implicated in a variety of neurological pathologies including stroke, head trauma, spinal cord injury, epilepsy, anxiety, and neurodegenerative diseases such as Alzheimer's Disease (Watkins and Collingridge, The NMDA Receptor, Oxford: IRL Press, 1989). A role for NMDA receptors in nociception and analgesia has been postulated as well (Dickenson, A cure for wind-up: NMDA receptor antagonists as potential analgesics. Trends Pharmacol. Sci. 11: 307, 1990). More recently, AMPA receptors have been widely studied for their possible contributions to such neurological pathologies (Fisher and Bogousslavsky, Evolving toward effective therapy for acute ischemic stroke. J. Amer. Med. Assoc. 270: 360, 1993; Yamaguchi et al., Anticonvulsant activity of AMPA/kainate antagonists: Comparison of GYKI 52466 and NBQX in maximal electroshock and chemoconvulsant seizure models. Epilepsy Res. 15: 179, 1993).
When activated by glutamate, the endogenous neurotransmitter, the NMDA receptor permits the influx of extracellular calcium (Ca.sup.2+) and sodium (Na.sup.+) through an associated ion channel. The NMDA receptor allows considerably more influx of Ca.sup.2+ than do kainate or AMPA receptors (but see below), and is an example of a receptor-operated Ca.sup.2+ channel. Normally, the channel is opened only briefly, allowing a localized and transient increase in the concentration of intracellular Ca.sup.2+ ([Ca.sup.2+ ].sub.i), which, in turn, alters the functional activity of the cell. However, prolonged increases in [Ca.sup.2+ ].sub.i, resulting from chronic stimulation of the NMDA receptor, are toxic to the cell and lead to cell death. The chronic elevation in [Ca.sup.2+ ].sub.i, resulting from stimulation of NMDA receptors, is said to be a primary cause of neuronal degeneration following a stroke (Choi, Glutamate neurotoxicity and diseases of the nervous system. Neuron 1: 623, 1988). Overstimulation of NMDA receptors is also said to be involved in the pathogenesis of some forms of epilepsy (Dingledine et al., Excitatory amino acid receptors in epilepsy. Trends Pharmacol. Sci. 11: 334, 1990), anxiety (Wiley and Balster, Preclinical evaluation of N-methyl-D-aspartate antagonists of antianxiety effects: A review. In: Multiple Sigma and PCP Receptor Ligands: Mechanisms for Neuromodulation and Neuroprotection? NPP Books, Ann Arbor, Mich., pp. 801-815, 1992), neurodegenerative diseases (Meldrum and Garthwaite, Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11: 379, 1990), and hyperalgesic states (Dickenson, A cure for wind-up: NMDA receptor antagonists as potential analgesics. Trends Pharmacol. Sci. 11: 307, 1990).
The activity of the NMDA receptor-ionophore complex is regulated by a variety of modulatory sites that can be targeted by selective antagonists. Competitive antagonists, such as the phosphonate AP5, act at the glutamate binding site, whereas noncompetitive antagonists, such as phencyclidine (PCP), MK-801 or magnesium (Mg.sup.2+), act within the associated ion channel (ionophore). There is also a glycine binding site that can be blocked selectively with compounds such as 7-chlorokynurenic acid. There is evidence suggesting that glycine acts as a co-agonist, so that both glutamate and glycine are necessary to fully elicit NMDA receptor-mediated responses. Other potential sites for modulation of NMDA receptor function include a zinc (Zn.sup.2+) binding site and a sigma ligand binding site. Additionally, endogenous polyamines such as spermine are believed to bind to a specific site and so potentiate NMDA receptor function (Ransom and Stec, Cooperative modulation of [.sup.3 H]MK-801 binding to the NMDA receptor-ion channel complex by glutamate, glycine and polyamines. J. Neurochem. 51: 830, 1988). the potentiating effect of polyamines on NMDA receptor function may be mediated via a specific receptor site for polyamines; polyamines demonstrating agonist, antagonist, and inverse agonist activity have been described (Reynolds, Arcaine is a competitive antagonist of the polyamine site on the NMDA receptor. Europ. J. Pharmacol. 177: 215, 1990; Williams et al., characterization of polyamines having agonist, antagonist, and inverse agonist effects at the polyamine recognition site of the NMDA receptor. Neuron 5: 199, 1990). Radioligand binding studies have demonstrated additionally that higher concentrations of polyamines inhibit NMDA receptor function (Reynolds and Miller, Ifenprodil is a novel type of NMDA receptor antagonist: Interaction with polyamines. Molec. Pharmacol. 36: 758, 1989; Williams et al., Effects of polyamines on the binding of [.sup.3 H] MK-801 to the NMDA receptor: Pharmacological evidence for the existence of a polyamine recognition site. Molec. Pharmacol. 36: 575, 1989; Sacaan and Johnson, Characterization of the stimulatory and inhibitory effects of polyamines on [.sup.3 H] TCP binding to the NMDA receptor-ionophore complex. Molec. Pharmacol. 37: 572, 1990). This inhibitory effect of polyamines on NMDA receptors is probably a nonspecific effect (i.e., not mediated via the polyamine receptor) because patch clamp electro-physiological studies have demonstrated that this inhibition is produced by compounds previously shown to act at the polyamine receptor as either agonists or antagonists (Donevan et al., Arcaine Blocks N-Methyl-D-Aspartate Receptor Responses by an Open Channel Mechanism: Whole-Cell and Single-Channel Recording Studies in Cultured Hippocampal Neurons. Molec. Pharmacol. 41: 727, 1992; Rock and Macdonald, Spermine and Related Polyamines Produce a Voltage-Dependent Reduction of NMDA Receptor Single-Channel Conductance. Molec. Pharmacol. 42: 157, 1992).
Recent studies have demonstrated the molecular diversity of glutamate receptors (reviewed by Nakanishi, Molecular Diversity of Glutamate Receptors and Implications for Brain Function. Science 258: 597, 1992). At least five distinct NMDA receptor subunits (NMDAR1 and NMDAR2A through NMDAR2D), each encoded by a distinct gene, have been identified to date. Also, in NMDAR1, alternative splicing gives rise to at least six additional isoforms. It appears that NMDAR1 is a necessary subunit, and that combination of NMDAR1 with different members of NMDAR2 forms the fully functional NMDA receptor-ionophore complex. The NMDA receptor-ionophore complex, thus, can be defined as a hetero-oligomeric structure composed of at least NMDAR1 and NMDAR2 subunits; the existence of additional, as yet undiscovered, subunits is not excluded by this definition. NMDAR1 has been shown to possess binding sites for glutamate, glycine, Mg.sup.2+, MK-801, and Zn.sup.2+. The binding sites for sigma ligands and polyamines have not yet been localized on NMDA receptor subunits, although ifenprodil recently has been reported to be more potent at the NMDAR2B subunit than at the NMDAR2A subunit (Williams, Ifenprodil discriminates subtypes of the N-methyl-D-aspartate receptor: selectivity and mechanisms at recombinant heteromeric receptors. Mol. Pharmacol. 44: 851, 1993).
Several distinct subtypes of AMPA and kainate receptors have been cloned as well (reviewed by Nakanishi, Molecular diversity of glutamate receptors and implications for brain function. Science 258: 597, 1992). Of particular relevance are the AMPA receptors designated GluR1, GluR2, GluR3, and GluR4 (also termed GluRA through GluRD), each of which exists in one of two forms, termed flip and flop, which arise by RNA alternative splicing. GluR1, GluR3 and GluR4, when expressed as homomeric and heteromeric receptors, are permeable to Ca.sup.2+, and are therefore examples of receptor-operated Ca.sup.2+ channels. Expression of GluR2 alone or in combination with the other subunits gives rise to a receptor which is largely impermeable to Ca.sup.2+. As most native AMPA receptors studied in situ are not Ca.sup.2+ -permeable (discussed above), it is believed that such receptors in situ possess at lest one GluR2 subunit.
Furthermore, it is hypothesized that the GluR2 subunit is functionally distinct by virtue of the fact that it contains an arginine residue within the putative pore-forming transmembrane region II; GluR1, GluR3 and GluR4 all contain a glutamine residue in this critical region (termed the Q/R site, where Q and R are the single letter designations for glutamine and arginine, respectively). The activity of the AMPA receptor is regulated by a number of modulatory sites that can be targeted by selective antagonists (Honore et al., Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists. Science 241: 701, 1988; Donevan and Rogawski, GYKI 52466, a 2,3-benzodiazepine, is a highly selective, noncompetitive antagonist of AMPA/kainate receptor responses. Neuron 10: 51, 1993). Competitive antagonists such as NBQX act at the glutamate binding site, whereas compounds such as GYKI 52466 appear to act noncompetitively at an associated allosteric site.
Compounds that act as competitive or noncompetitive antagonists at the NMDA receptor are said to be effective in preventing neuronal cell death in various in vitro neurotoxicity assays (Meldrum and Garthwaite, Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11: 379, 1990) and in in vivo models of stroke (Scatton, Therapeutic potential of NMDA receptor antagonists in ischemic cerebrovascular disease in Drug Strategies in the Prevention and Treatment of Stroke, IBC Technical Services Ltd., 1990). Such compounds are also effective anticonvulsants (Meldrum, Excitatory amino acid neurotransmission in epilepsy and anticonvulsant therapy in Excitatory Amino Acids. Meldrum, Moroni, Simon, and Woods (Eds.), New York; Raven Press, p. 655, 1991), anxiolytics (Wiley and Balster, Preclinical evaluation of N-methyl-D-aspartate antagonists for antianxiety effects: A review. In: Multiple Sigma and PCP Receptor Ligands: Mechanisms for Neuromodulation and Neuroprotection? NPP Books, Ann Arbor, Mich., pp. 801-815, 1992), and analgesics (Dickenson, A cure for wind-up: NMDA receptor antagonists as potential analgesics. Trends Pharmacol. Sci. 11: 307, 1990), and certain NMDA receptor antagonists may lessen dementia associated with Alzheimer's Disease (Hughes, Merz' novel approach to the treatment of dementia. Script No. 1666: 24, 1991).
Similarly, AMPA receptor antagonists have come under intense scrutiny as potential therapeutic agents for the treatment of such neurological disorders and diseases. AMPA receptor antagonists have been shown to possess neuroprotectant (Fisher and Bogousslavsky, Evolving toward effective therapy for acute ischemic stroke. J. Amer. Med. Assoc. 270: 360, 1993) and anticonvulsant (Yamaguchi et al., Anticonvulsant activity of AMPA/kainate antagonists: comparison of GYKI 52466 and NBQX in maximal electroshock and chemoconvulsant seizure models. Epilepsy Res. 15: 179, 1993) activity in animal models of ischemic stroke and epilepsy, respectively.
The nicotinic cholinergic receptor present in the mammalian CNS is another example of a receptor-operated Ca.sup.2+ channel (Deneris et al., Pharmacological and functional diversity of neuronal nicotinic acetylcholine receptors. Trends Pharmacol. Sci. 12: 34, 1991). Several distinct receptor subunits have been cloned, and these subunits can be expressed, in Xenopus oocytes for example, to form functional receptors with their associated cation channels. It is hypothesized that such receptor-ionophore complexes are heteropentameric structures. The possible role of nicotinic receptor-operated Ca.sup.2+ channels in the pathology of neurological disorders and diseases such as ischemic stroke, epilepsy and neurodegenerative diseases has been largely unexplored.
It has been demonstrated previously that certain spider and wasp venoms contain arylalkylamine toxins (also called polyamine toxins, arylamine toxins, acylpolyamine toxins or polyamine amide toxins) with activity against glutamate receptors in the mammalian CNS (for reviews see Jackson and Usherwood, Spider toxins as tools for dissecting elements of excitatory amino acid transmission. Trends Neurosci. 11: 278, 1988; Jackson and Parks, Spider Toxins: Recent Applications In Neurobiology. Annu. Rev. Neurosci. 12: 405, 1989; Saccomano et al., Polyamine spider toxins: Unique pharmacological tools. Annu. Rep. Med. Chem. 24: 287, 1989; Usherwood and Blagbrough, Spider Toxins Affecting Glutamate Receptors: Polyamines in Therapeutic Neurochemistry. Pharmacol. Therap. 52: 245, 1991; Kawai, Neuroactive Toxins of Spider Venoms. J. Toxicol. Toxin Rev. 10: 131, 1991). Arylalkylamine toxins were initially reported to be selective antagonists of the AMPA/kainate subtypes of glutamate receptors in the mammalian CNS (Kawai et al., Effect of a spider toxin on glutaminergic synapses in the mammalian brain. Biomed. Res. 3: 353, 1982, Saito et al., Spider Toxin (JSTX) blocks glutamate synapse in hippocampal pyramidal neurons. Brain Res. 346: 397, 1985; Saito et al., Effects of a spider toxin (JSTX) on hippocampal CA1 neurons in vitro. Brain Res. 481: 16, 1989; Akaike et al., Spider toxin blocks excitatory amino acid responses in isolated hippocampal pyramidal neurons. Neurosci. Lett. 79: 326, 1987; Ashe et al., Argiotoxin-636 blocks excitatory synaptic transmission in rat hippocampal CA1 pyramidal neurons. Brain Res. 480: 234, 1989; Jones et al., Philanthotoxin blocks quisqualate-induced, AMPA-induced and kainate-induced, but not NMDA-induced excitation of rat brainstem neurones in vivo. Br. J. Pharmacol. 101: 968, 1990). Subsequent studies have demonstrated that while certain aryalkylamine toxins are both nonpotent and nonselective at various glutamate receptors, other arylalkylamines are both very potent and selective at antagonizing responses mediated by NMDA receptor activation in the mammalian CNS (Mueller et al., Effects of polyamine spider toxins on NMDA receptor-mediated transmission in rat hippocampus in vitro. Soc. Neurosci. Abst. 15: 945, 1989; Mueller et al., Arylamine spider toxins antagonize NMDA receptor-mediated synaptic transmission in rat hippocampal slices. Synapse 9: 244, 1991; Parks et al., Polyamine spider toxins block NMDA receptor-mediated increases in cytosolic calcium in cerebellar granule neurons. Soc. Neurosci. Abst. 15: 1169, 1989; Parks et al., Arylamine toxins from funnel-web spider (Agelenopsis aperta) venom antagonize N-methyl-D-aspartate receptor function in mammalian brain. J. Biol. Chem. 266: 21523, 1991; Priestley et al., Antagonism of responses to excitatory amino acids on rat cortical neurones by the spider toxin, argiotoxin-636. Br. J. Pharmacol. 97: 1315, 1989; Draguhn et al., Argiotoxin-636 inhibits NMDA-activated ion channels expressed in Xenopus oocytes. Neurosci. Lett. 132: 187, 1991; Kiskin et al., A highly potent and selective N-methyl-D-aspartate receptor antagonist from the venom of the Agelenopsis aperta spider. Neuroscience 51: 11, 1992; Brackley et al., Selective antagonism of native and cloned kainate and NMDA receptors by polyamine-containing toxins. J. Pharmacol. Exptl. Therap. 266: 1573, 1993; Williams, Effects of Agelenopsis aperta toxins on the N-methyl-D-aspartate receptor: Polyamine-like and high-affinity antagonist actions. J. Pharmacol. Exptl. Therap. 266: 231, 1993). Inhibition of nicotinic cholinergic receptors by the arylalkylamine toxin philanthotoxin has also been reported (Rozetal et al., Allosteric inhibition of nicotinic acetylcholine receptors of vertebrates and insects by philanthotoxin. J. Pharmacol. Exptl. Therap. 249: 123, 1989).
Parks et al. (Arylamine toxins from funnel-web spider (Agelenopsis aperta) venom antagonize N-methyl-D-aspartate receptor function in mammalian brain. J. Biol. Chem. 266: 21523, 1991), describe arylalkylamine spider toxins (.alpha.-agatoxins) which antagonize NMDA receptor function in mammalian brain. The authors discuss the mechanism of action of arylalkylamine toxins, and indicate that an NMDA receptor-operated ion channel is the probable site of action of the .alpha.-agatoxins, and most probably other spider venom arylalkylamines. They state:
The discovery that endogenous polyamines in the vertebrate brain modulate the function of NMDA receptors suggests that the arylamine toxins may produce their antagonism via a polyamine-binding site on glutamate receptors. Brackley et al. studied the effects of spermine and philanthotoxin 433 on the responses evoked by application of excitatory amino acids in Xenopus oocytes injected with mRNA from rat or chick brain. These authors reported that, at concentrations below those that antagonize glutamate receptor function, both spermine and philanthotoxin potentiate the effects of excitatory amino acids and some other neurotransmitters. On the basis of these and other data, Brackley et al. concluded that the arylamine toxins may, by binding nonspecifically to the membranes of excitable cells, reduce membrane fluidity and alter receptor function. The validity of this intriguing idea for NMDA receptor function is not well supported by two recent binding studies. Reynolds reported that argiotoxin 636 inhibits the binding of [.sup.3 H] MK-801 to rat brain membranes in a manner that is insensitive to glutamate, glycine, or spermidine. This author concluded that argiotoxin 636 exerts a novel inhibitor effect on the NMDA receptor complex by binding to one of the Mg.sup.2+ sites located within the NMDA-gated ion channel. Binding data reported by Williams et al. also support the conclusion that argiotoxin 636 does not act primarily at the polyamine modulatory site on the NMDA receptor, but rather acts directly to produce an activity-dependent block of the ion channel. It is already known that compounds such as phencyclidine and ketamine can block the ion channels associated with both arthropod muscle glutamate receptors and mammalian NMDA receptors. Thus, it seems possible that vertebrate and invertebrate glutamate receptors share additional binding sites for allosteric modulators of receptor function, perhaps related to divalent cation-binding sites. Clearly, considerable additional work will be needed to determine if the arylamines define such a novel regulatory site. PA1 [.sup.125 I]Arg-636 bound to rat forebrain membranes with K.sub.d and B.sub.max values of 11.25 .mu.M and 28.95 pmol/mg protein (80% specific). The ability of other known polyamines and recently discovered polyamines from Agelenopsis aperta to inhibit binding paralleled neuroactivity as functional NMDA antagonists. No other compounds tested were able to block specific binding. PA1 Although .alpha.-agatoxins can interact at the positive allosteric polyamine site on the NMDA receptor, stimulatory effects produced by this interaction may be masked in functional assays due to a separate action of the toxins as high-affinity, noncompetitive antagonists of the receptor. PA1 R.sup.1, R.sup.2, R.sup.5, and R.sup.6 and --H; PA1 or R.sup.2 is methyl, and R.sup.1, R.sup.5, and R.sup.6 are --H; PA1 or R.sup.1 is methyl, and R.sup.2, R.sup.5, and R.sup.6 are --H.
Usherwood and Blagbrough (Spider Toxins Affecting Glutamate Receptors: Polyamines in Therapeutic Neurochemistry. Pharmacol. Therap. 52: 245, 1991) describe a proposed intracellular binding site for arylalkylamine toxins (polyamine amide toxins) located within the membrane potential field referred to as the QUIS-R channel selectivity filter. The authors postulate that the binding site for polyamine amide toxins may occur close to the internal entrance of the channel gated by the QUIS-R of locust muscle. The authors also note that one such toxin, argiotoxin-636, selectively antagonizes the NMDA receptor in cultured rat cortical neurons.
Gullak et al. (CNS binding sites of the novel NMDA antagonist Arg-636. Soc. Neurosci. Abst. 15: 1168, 1989), describe argiotoxin-636 (Arg-636) as a polyamine (arylalkylamine) toxin component of a spider venom. This toxin is said to block NMDA-induced elevation of cGMP in a noncompetitive fashion. The authors state that:
The authors then stated that polyamines (arylalkylamines) may antagonize responses to NMDA by interacting with membrane ion channels.
Seymour and Mena (In vivo NMDA antagonist activity of the polyamine spider venom component, argiotoxin-636. Soc. Neurosci. Abst. 15: 1168, 1989) describe studies that are said to show that argiotoxin-636 does not significantly affect locomotor activity at doses that are effective against audiogenic seizures in DBA/2 mice, and that it significantly antagonizes NMDA-induced seizures with a minimal effective dose of 32 mg/kg given subcutaneously (s.c.).
Herold and Yaksh (Anesthesia and muscle relaxation with intrathecal injections of AR636 and AG489, two acylpolyamine spider toxins, in rats. Anesthesiology 77: 507, 1992) describe studies that are said to show that the arylalkylamine argiotoxin-636 (AR636), but not agatoxin-489 (AG489), produces muscle relaxation and anesthesia following intrathecal administration in rats.
Williams (Effects of Agelenopsis aperta toxins on the N-methyl-D-aspartate receptor: Polyamine-like and high-affinity antagonist actions, J. Pharmacol. Exptl. Therap. 266: 231, 1993) reports that the .alpha.-agatoxins (arylalkylamines) Agel-489 and Agel-505 enhance the binding of [.sup.3 H]MK-801 to NMDA receptors on membranes prepared from rat brain by an action at the stimulatory polyamine receptor; polyamine receptor agonists occluded the stimulatory effects of Agel-489 and Agel-505 and polyamine receptor antagonists inhibited the stimulatory effect of Agel-505. Higher concentrations of Agel-489 and Agel-505, and argiotoxin-636 at all concentrations tested, had inhibitory effects on the binding of [.sup.3 H]MK-801. In Xenopus oocytes voltage-clamped at -70 mV, Agel-505 inhibited responses to NMDA with an IC.sub.50 of 13 nM; this effect of Agel-505 occurred at concentrations approximately 10,000-fold lower than those that affected [.sup.3 H]MK-801 binding. Responses to kainate were inhibited only 11% by 30 nM Agel-505. The antagonism of NMDA-induced currents by Agel-505 was strongly voltage-dependent, consistent with an open-channel blocking effect of the toxin. Williams states:
Brackley et al. (Selective antagonism of native and cloned kainate and NMDA receptors by polyamine-containing toxins, J. Pharmacol. Exp. Therap. 266: 1573, 1993) report that the polyamine-containing toxins (arylalkylamines) philanthotoxin-343 (PhTX-343) and argiotoxin-636 (Arg-636) produce reversible, noncompetitive, partly voltage-dependent antagonism of kainate- and NMDA-induced currents in Xenopus oocytes injected with rat brain RNA. Arg-636 was demonstrated to be selective for NMDA-induced responses (IC.sub.50 =0.04 .mu.M) compared to kainate-induced responses (IC.sub.50 =0.07 .mu.M), while PhTX-343 was selective for kainate-induced responses (IC.sub.50 =0.12 .mu.M) compared to NMDA-induced responses (IC.sub.50 =2.5 .mu.M). Arg-636 more potently antagonized responses to NMDA in Xenopus oocytes expressing cloned NMDAR1 subunits (IC.sub.50 =0.09 .mu.M) than responses to kainate in oocytes expressing either cloned GluR1 (IC.sub.50 =3.4 .mu.M) or GluR1+GluR2 subunits (IC.sub.50 =300 .mu.M). PhTX-343, on the other hand, was equipotent at antagonizing NMDAR1 (IC.sub.50 =2.19 .mu.M) and GluR1 (IC.sub.50 =2.8 .mu.M), but much less potent against GluR1+GluR2 subunits (IC.sub.50 =270 .mu.M).
Raditsch et al. (Subunit-specific block of cloned NMDA receptors by argiotoxin-636. FEBS Lett. 324: 63, 1993) report that Arg-636 more potently antagonizes responses in Xenopus oocytes expressing NMDAR1+NMDAR2A subunits (IC.sub.50 =9 nM) or NMDAR1+NMDAR2B subunits (IC.sub.50 =2.5 nM) than NMDAR1+NMDAR2C subunits (IC.sub.50 =460 nM), even though all of the receptor subunits contain an asparagine residue in the putative pore-forming transmembrane region II (the Q/R site, as discussed above). The authors state that the large difference in Arg-636 sensitivity between NMDAR1+NMDAR2A and NMDAR1+NMDAR2C channels "must be conferred by other structural determinants."
Herlitz et al. (Argiotoxin detects molecular differences in AMPA receptor channels. Neuron 10: 1131, 1993) report that Arg-636 antagonizes subtypes of AMPA receptors in a voltage- and use-dependent manner consistent with open-channel blockade. Arg-636 potently antagonizes Ca.sup.2+ -permeable AMPA receptors comprised of GluRAi (K.sub.i =0.35 .mu.M), GluRCi (K.sub.i =0.23 .mu.M), or GluRDi subunits (K.sub.i =0.43 .mu.M), while being essentially ineffective against Ca.sup.2+ -impermeable GluRBi subunits at concentrations up to 10 .mu.M.
Other data reported by these investigators strongly suggest that the Q/R site in the putative pore-forming transmembrane region II is of primary importance in determining Arg-636 potency and Ca.sup.2+ permeability.
Blaschke et al. (A single amino acid determines the subunit-specific spider toxin block of .alpha.-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor channels. Proc. Natl. Acad. Sci. USA 90: 6528, 1993) report that the arylalkylamine JSTX-3 potently antagonizes responses to kainate in Xenopus oocytes expressing GluR1 (IC.sub.50 =0.04 .mu.M) or GluR3 (IC.sub.50 =0.03 .mu.M) subunits, but that expressed receptors in which a GluR2 subunit is present are essentially unaffected by the toxin. Site-directed mutagensis studies strongly implicate the Q/R site as the primary site influencing toxin potency.
Nakanishi et al. (Bioorganic studies of transmitter receptors with philanthotoxin analogs. Pure Appl. Chem., in press) have synthesized a number of highly potent photoaffinity labeled philanthotoxin (PhTX) analogs. Such analogs have been studied on expressed nicotinic cholinergic receptors as a model system for receptor-operated calcium channels. These investigators suggest that these PhTX analogs block the ion channel with the hydrophobic headpiece of the toxin binding to a site near the cytoplasmic surface while the polyamine tail extends into the ion channel from the cytoplasmic side.